What Breast Medical Oncologists Need From Pathologists

Updated: Oct 28, 2013
  • Author: Lyndsay Harris, MD, FRCPC; Chief Editor: D Craig Allred, MD  more...
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Overview

Overview

Breast medical oncologists use the information provided by pathologists to make decisions about systemic therapy. The image below provides a schematic representation of the decision tree used by medical oncologists when deciding whether and which systemic therapy to recommend to a patient.

The flow chart shows the idealized decision making The flow chart shows the idealized decision making process of the medical oncologist when deciding whether and what systemic therapy to give. Remember that the decision to give a specific systemic therapy to an individual patient is based on the expected benefit as compared with the possible risks in each individual patient. The blue text provides links to the text below explaining the role of the pathologist in providing the necessary information to make these decisions. * The cut off is 2 cm for pure mucinous carcinomas. **OncotypeDX can help decisions in patients with tumors between 1 and 2 cm.

Providing an easy-to-read summary or synoptic report, including the key prognostic and predictive factors, is particularly helpful to the medical oncologist. Although many of the pathology elements are continuous variables, clinical decisions are not. Being aware of the critical factors that influence decisions in a particular patient when evaluating a specimen is important for pathologists. Medical oncologists should be knowledgeable about sources of controversy and should look for potential pitfalls in the pathology report. Tumor size, grade, axillary lymph node status, steroid hormone receptor status, and HER2/neu overexpression are the factors that most commonly influence treatment decisions and are discussed in detail below.

In 2007, the American Society of Clinical Oncology (ASCO) updated the recommendations for the use of tumor marker tests in the prevention, screening, treatment, and surveillance of breast cancer. An overview of the 2007 ASCO recommendations is presented in Table 1. [1]

Table 1. Overview of the 2007 ASCO Guidelines for the Use of Tumor Markers in the Prevention, Screening, Treatment, and Surveillance of Breast Cancer (Open Table in a new window)

Marker Recommendation
CA 15-3 and CA 27.29 as markers for breast cancer as screening, diagnostic, or staging tests Present data are insufficient to recommend CA 15-3 or CA 27.29 for screening, diagnosis, and staging.
CA 15-3 and CA 27.29 to detect recurrence after primary breast cancer therapy Present data do not support the use of CA 15-3 and CA 27.29 for monitoring patients for recurrence after primary breast cancer therapy.
CA 15-3 and CA 27.29 to contribute to decisions regarding therapy for metastatic breast cancer For monitoring patients with metastatic disease during active therapy, CA 27.29 or CA 15-3 can be used in conjunction with diagnostic imaging, history, and physical examination. Present data are insufficient to recommend use of CA 15-3 or CA 27.29 alone for monitoring response to treatment. However, in the absence of readily measurable disease, an increasing CA 15-3 or CA 27.29 level may be used to indicate treatment failure. Caution should be used when interpreting a rising CA 27.29 or CA 15-3 level during the first 4-6 weeks of a new therapy, since spurious early rises may occur.
CEA for screening, diagnosis, staging, or routine surveillance of breast cancer after primary therapy CEA is not recommended for screening, diagnosis, staging, or routine surveillance of breast cancer after primary therapy.
CEA to contribute to decisions regarding therapy for metastatic breast cancer For monitoring patients with metastatic disease during active therapy, CEA can be used in conjunction with diagnostic imaging, history, and physical examination. Present data are insufficient to recommend use of CEA alone for monitoring response to treatment. However, in the absence of readily measurable disease, an increasing CEA level may be used to indicate treatment failure. Caution should be used when interpreting a rising CEA level during the first 4-6 weeks of a new therapy, since spurious early rises may occur. There is no change from the guideline published in 2000.
ERs and PgRs ER and PgR should be measured in every primary invasive breast cancer and may be measured on metastatic lesions if the results would influence treatment planning. In both premenopausal and postmenopausal patients, steroid hormone receptor status should be used to identify patients most likely to benefit from endocrine forms of therapy in both early breast cancer and metastatic disease. In patients with DCIS who are candidates for hormonal therapy, data are insufficient to recommend routine measurement of ER and PgR for therapy recommendations.
DNA flow cytometry–based parameters Present data are insufficient to recommend use of DNA content, S phase, or other flow cytometry–based markers of proliferation to assign patients to prognostic groups.
Immunohistochemically based markers of proliferation Present data are insufficient to recommend measurement of Ki67, cyclin D, cyclin E, p27, p21, thymidine kinase, topoisomerase II, or other markers of proliferation to assign patients to prognostic groups.
HER2 evaluation in breast cancer HER2 expression and/or amplification should be evaluated in every primary invasive breast cancer, either at the time of diagnosis or at the time of recurrence, principally to guide selection of trastuzumab in the adjuvant and/or metastatic setting. Other utilities for HER2 evaluation are also discussed separately below.
HER2 to define prognosis for early-stage breast cancer in the absence of systemic therapy HER2 amplification, HER2 overexpression, and the presence of HER2 extracellular domain are generally associated with a poorer prognosis. However, the value of this information in clinical practice is questionable, and the use of HER2 for determining prognosis is not recommended.
HER2 to select patients for anti-HER2–based therapy High levels of tissue HER2 expression or HER2 gene amplification should be used to identify patients for whom trastuzumab may be of benefit for treatment of breast cancer in the adjuvant or metastatic disease settings.
The utility of HER2 for predicting response to specific chemotherapeutic agents level II evidence (prospective therapeutic trials in which marker utility is a secondary study objective) suggests that overexpression of HER2 (3+ by protein or >2.0 FISH ratio by gene amplification) identifies patients who would have a greater benefit from anthracycline-based adjuvant therapy. If a clinician is considering chemotherapy for a patient with HER2-positive breast cancer, it is recommended that an anthracycline be strongly considered in the absence of contraindications to anthracycline therapy. In the context of trastuzumab therapy, there is level I evidence (single, high-powered, prospective, randomized, controlled trials specifically designed to test the marker or a meta-analyses of well-designed studies) that a nonanthracycline regimen may produce similar outcomes. At present, the Update Committee does not recommend that HER2 be used to guide use of taxane chemotherapy in the adjuvant setting.
HER2 to determine sensitivity to endocrine therapy HER2 should not be used to withhold endocrine therapy for a patient with hormone receptor–positive breast cancer, nor should it be used to select one specific type of endocrine therapy over another.
Utility of circulating extracellular domain of HER2 Measuring circulating extracellular domain of HER2 is not currently recommended for any clinical setting.
p53 as a marker for breast cancer Present data are insufficient to recommend p53 measurements for management of patients with breast cancer.
uPA and PAI-1 as a marker for breast cancer (Note: This topic is new to the guideline) uPA/PAI-1 measured by ELISA on a minimum of 300 mg of fresh or frozen breast cancer tissue may be used to determine prognosis in patients with newly diagnosed node-negative breast cancer. IHC for these markers is not accurate, and the prognostic value of ELISA using smaller tissue specimens has not been validated. Low levels of both markers are associated with a sufficiently low risk of recurrence, especially in hormone receptor–positive women who will receive adjuvant endocrine therapy, so chemotherapy will only contribute minimal additional benefit. Furthermore, CMF-based adjuvant chemotherapy provides substantial benefit compared with observation alone in patients with high risk of recurrence as determined by high levels of uPA and PAI-1.
Cathepsin D as a marker for breast cancer Present data are insufficient to recommend use of cathepsin D measurements for management of patients with breast cancer.
Cyclin E fragments as markers for breast cancer (Note: This topic is new to the guideline) Present data are insufficient to recommend use of whole-length or fragment measurements of cyclin E for management of patients with breast cancer.
Proteomic analysis for breast cancer (Note: This topic is new to the guideline) Present data are insufficient to recommend use of proteomic patterns for management of patients with breast cancer.
Multiparameter gene expression analysis for breast cancer (Note: This topic is new to the guideline) In newly diagnosed patients with node-negative, ER-positive breast cancer, the Oncotype DX assay can be used to predict the risk of recurrence in patients treated with tamoxifen. Oncotype DX may be used to identify patients who are predicted to obtain the most therapeutic benefit from adjuvant tamoxifen and may not require adjuvant chemotherapy. In addition, patients with high recurrence scores appear to achieve relatively more benefit from adjuvant chemotherapy (specifically (C)MF) than from tamoxifen. There are insufficient data at present to comment on whether these conclusions generalize to hormonal therapies other than tamoxifen or whether this assay applies to other chemotherapy regimens. The precise clinical utility and appropriate application for other multiparameter assays, such as the MammaPrint assay, the "Rotterdam Signature," and the Breast Cancer Gene Expression Ratio are under investigation.
Bone marrow micrometastases as markers for breast cancer (Note: This topic is new to the guideline) Present data are insufficient to recommend assessment of bone marrow micrometastases for management of patients with breast cancer.
Circulating tumor cell assays as markers for breast cancer (Note: This topic is new to the guideline) The measurement of CTCs should not be used to diagnose breast cancer or to influence any treatment decisions in patients with breast cancer. Similarly, the use of the recently FDA-cleared test for CTC (CellSearch assay) in patients with metastatic breast cancer cannot be recommended until further validation confirms the clinical value of this test.
Abbreviations: CEA, carcinoembryonic antigen; CMF, cyclophosphamide, methotrexate, and fluorouracil; CTCs, circulating tumor cells; DCIS, ductal carcinoma in situ; ELISA, enzyme-linked immunosorbent assay; ER, estrogen receptor; FDA, US Food and Drug Administration; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; PAI-1, plasminogen activator inhibitor 1; PgR, progesterone receptor; uPA, urokinase plasminogen activator
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Tumor Size

Definition

Tumor size is measured as the largest dimension of invasive tumor only, in centimeters.

Practical considerations

When available, the pathologic tumor size takes precedence over the clinical tumor size. The tumor size reported in the pathology report should reflect the pathologist’s best assessment of the largest dimension of invasive tumor only. This is usually the microscopic invasive tumor size, but, in many circumstances, correlation with findings from gross examination, prior specimens, different imaging modalities, and clinical information is needed to most accurately estimate the tumor size. Benign and in situ mass-forming lesions, biopsy site changes, fixation, and processing can all affect tumor size measurements.

Tumor size should be measured with a precision of 1 mm. Tumor size cutoffs of 1 cm and of 2 cm are particularly important for treatment decisions. A statement about how the measurement was obtained or estimated is helpful. (For example: microscopic, gross, largest dimension in prior core biopsy is…, estimated by…, at least…, likely not more than…, correlation with imaging and clinical findings is needed.)

Controversies

Multiple tumor foci are measured separately. A comment about their relative location and the intervening breast tissue is needed. The size of the largest focus is used for staging. [2]

In patients who have undergone preoperative chemotherapy, the presence of residual invasive and in situ disease in the tumor bed and lymph nodes provides important information. However, at this time, the only validated pathologic measure is pathologic complete response (ie, lack of invasive disease at surgery), in addition to the size of the residual tumor. Cellularity of the residual tumor is also emerging as an important prognostic indicator. Classification of response either by Miller-Payne scoring or Residual Cancer Burden scoring appear to predict clinical outcome. [3] The latter scoring systems require additional validation for widespread use in clinical practice but may provide useful information for the oncologist.

After neoadjuvant chemotherapy, tumors with a partial response often present as scattered foci of tumor with variable cellularity with a residual tumor bed that can be identified as reactive changes, including fibrosis, histiocytes, and fat necrosis, but is sometimes microscopically indistinguishable from normal breast tissue. Tumor size after preoperative chemotherapy should include these individual foci within the original tumor bed within a single measurement. The Miller-Payne score divides the reduction in overall cellularity in 5 grades, with grade 1 as no significant reduction in overall cellularity and grade 5 as a complete pathologic response at the site of the primary tumor. Residual Cancer Burden scoring [4] combines the size of the primary tumor bed in 2 dimensions, the cellularity of the invasive cancer and the amount of residual disease in the lymph nodes (both number and size of largest focus are needed to calculate the score) into a single score.

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Histologic Grade

Histologic grade reflects how far the tumor architecture and cytology deviate from normal and how rapidly the tumor proliferates. The College of American Pathologists (CAP) recommends the use of the Nottingham combined histologic grade, also called the modified Scarff-Bloom-Richardson grading system (see Table 2).

Table 2. The Nottingham Combined Histologic Grade, Also Called the Modified Scarff-Bloom-Richardson Grade (Open Table in a new window)

Feature   Score
Tubule formation >75% 1
10-75% 2
< 10% 3
Nuclear pleomorphism Small, regular uniform cells 1
Moderate increase in size and variability 2
Marked variation 3
Mitotic counts per 10 HPF (field diameter, 0.59 mm) 0-9 1
10-19 2
>20 3
Scoring
3-5 points Well differentiated Grade 1
6-7 points Moderately differentiated Grade 2
8-9 points Poorly differentiated Grade 3

Practical considerations

The size of the HPF (40x) needs to be taken into account when assessing mitotic counts. Mitotic counts should be assessed at the periphery of the tumor, in the most mitotically active area. One should first look around at low power to look for mitotic figures. Only definite mitotic figures should be counted.

Controversies

Assessment of tubule formation and mitotic count on core biopsy may not be representative of the entire tumor. When more than the nuclear grade is reported on the core biopsy, a possible change in grade on the excision specimen should be expected.

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Axillary Lymph Node Status

Definitions

See Table 3. [2]

Table 3. Definitions of Terms Used to Describe Axillary Lymph Node Metastases (Open Table in a new window)

  Definition Pathologic N Stage
Isolated tumor cells Tumor cell deposit ≤0.2 mm pN0i+
Micrometastasis Tumor cell deposit >0.2 mm and ≤2 mm pN1mic
Macrometastasis Tumor cell deposit >2 mm pN1
Number of positive axillary



lymph nodes



If there is one tumor cell deposit >2 mm, then all lymph nodes with micrometastases or macrometastases are counted as positive lymph nodes. Cancerous nodules in the axillary fat without histologic evidence of residual lymph node tissue are counted as positive lymph nodes. pN1a (1-3 positive axillary lymph nodes)



pN2a (4-9 positive axillary lymph nodes)



pN3a (≥10 positive axillary lymph nodes)



Practical considerations

Although no consensus on how to process lymph nodes to maximize the detection of isolated tumor cells (ITCs) and micrometastases exists, pathologic evaluation of axillary lymph nodes should identify all macrometastases (>2 mm). This requires submitting grossly negative lymph nodes entirely sectioned at 2-mm intervals and examining at least one hematoxylin and eosin (H&E)–stained section of each block.

Because the number of positive axillary lymph nodes is taken into consideration for pN staging of breast carcinoma, counting the number of positive lymph nodes is necessary. Therefore, each separate lymph node must be identified in a separate cassette or by inking. A representative section of each grossly positive lymph node is sufficient. Cytokeratin immunostains and ER immunostains (for ER-positive tumors) can be helpful to confirm the presence of tumor cell deposits, especially for lobular carcinomas or carcinomas with a histiocytoid appearance. The presence of extracapsular extension should be mentioned in the pathology report, as this has both local and systemic recurrence implications.

Controversies

Protocols used to increase the likelihood of identifying micrometastases and ITC vary widely from laboratory to laboratory. Techniques used include step sectioning at different intervals through the block and cytokeratin immunohistochemical stains. When interpreting immunohistochemical stains, correlation with H&E and morphologic features (particularly nuclear features) is needed. Only cytokeratin-positive tumor cells (morphologically identifiable as such by comparison with H&E, by cell size and nuclear morphology), not cytokeratin-positive cells, are reported as ITC. It is helpful to state how the lymph nodes were examined (eg, number of H&E levels, cytokeratin immunostain).

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Tissue-Based Tumor Markers

Indications for testing

In 2007, ASCO updated the recommendations for the use of tumor marker tests in the prevention, screening, treatment, and surveillance of breast cancer. An overview of the 2007 ASCO recommendations is presented in Table 1.

Practical considerations

For the results of any prognostic/predictive test to be clinically meaningful, rigorous quality-control measures must be applied and followed. Any new test should be compared with a reference test for which there has been clinical validation. Clinical validation requires a study demonstrating that the test predicts clinical outcome.

When formalin-fixed paraffin-embedded (FFPE) tissue is used for testing, the importance of appropriate fixation cannot be overemphasized. Ten percent neutral buffered formalin is the standard fixative. Fixing within one hour of removal from the patient is recommended. Specimens should be sliced into 0.5- to 1-cm sections prior to fixing and placed in sufficient volumes of formalin. Duration of fixation should be kept between 6 and 48 hours. Sections for processing should be 2-4 mm thick.

Controversies

Excellent concordance for HER2 and ER testing on core biopsy and excision specimens has been reported. [5] Adequate fixation appears to be a more important factor than biologic tumor heterogeneity. ER and HER2 testing is preferably performed on core biopsy specimens in the authors’ practice because it is easier to standardize fixation times for core biopsy specimens and because test results are available at an earlier step in the management of the patient.

Studies addressing the utility of testing multiple tumors in the same patient and testing the initial biopsy and the resection specimen after neoadjuvant chemotherapy are needed. It is not routine to test multiple foci of disease or before and after neoadjuvant chemotherapy, although these recommendations may change in the future with emerging data.

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Estrogen Receptor and Progesterone Receptor Status

Indications for testing

The result of ER and PgR testing is used to identify patients who are most likely to benefit from endocrine therapy (tamoxifen, ovarian ablation, aromatase inhibitors, irreversible ER inhibitors). ER and PgR should be measured in every primary invasive breast cancer and may be measured on metastatic lesions if the results would influence treatment planning. In both premenopausal and postmenopausal patients, steroid hormone receptor status should be used to identify patients most likely to benefit from endocrine forms of therapy in both the early breast cancer and metastatic disease settings. In patients with DCIS who are candidates for hormonal therapy, data are insufficient to recommend routine measurement of ER and PgR for therapy recommendations. [1]

Practical considerations

Currently preferred method of testing

IHC is now the method of choice in pathology laboratories.

Three ER antibodies are frequently used (1D5, Dakocytomation, Dako, Carpenteria, CA; 6F11, Novocastra Laboratories Ltd, Novocastra, Newcastle upon Tyne, UK; SP1, Lab-Vision-NeoMarkers, Neomarkers, Fremont, CA).

Heat-induced epitope retrieval times/techniques and optimal dilutions must be determined by individual laboratories.

Interpretation and reporting

The percentage or proportion of cells expressing the antigen should be specified in the pathology report. [6] ER and PR are nuclear stains. To decide to use hormonal therapy, any ER staining is considered relevant. The term low-positive can be used when the proportion of cells staining is between 1% and 9%.

Scoring systems incorporating both intensity of staining and proportion of cells staining such as the “Allred score” have been correlated with response to therapy. [7] There is currently no generally accepted scoring system. The pathologist plays an important role in avoiding false-negative results (see below).

Quality concerns [5]

The most important concern in testing for ER is false-negative results. These can result from fixation times that are too short (< 6 h) [8] or too long (weeks), [9] decalcification and mercury-containing fixatives (B5), storage of unstained sections for more than 1 week, and insufficient antigen retrieval.

Insufficient antigen retrieval is thought to be one of the most important factors. [10] Table 4 gives a number of practical tips to avoid false negative results.

Avoiding false-negative estrogen receptor and progesterone receptor immunohistochemistry results

Considerations concerning pre-analytic/analytic variables are as follows:

  • Appropriate fixation: Time from tissue acquisition to fixation should be as short as possible; specimens should be fixed in neutral buffered formalin for 6-48 hours; they should be sliced at 5- to 10-mm intervals and placed in sufficient volume of neutral buffered formalin; time to fixation and duration of fixation should be recorded for each sample
  • Appropriate antigen retrieval (to be determined by individual laboratories, at least 25 minutes has been suggested)
  • Appropriate controls (ideally, controls with both high and low levels of ER should be used)

Considerations during pathologist interpretation are as follows:

  • When possible, the test should be performed on a block with an internal control because this internal control will be an indicator of fixation issues; normal benign ducts and lobules show patchy to diffuse ER immunopositivity
  • ER positivity is expected in virtually all grade 1 tumors, in pure tubular carcinomas, in colloid carcinomas, and in classic lobular carcinoma. [11] Negative results in these tumors should be questioned with repeat testing, if indicated; ER-negative, PR-positive tumors are very uncommon (< 5%); the test should be repeated, if indicated
  • If the results on core biopsy are questioned, repeat testing on an excision is warranted
  • Negative results on unstained sections stored for prolonged periods should be questioned; unstained sections can be stored in a freezer to slow down oxidation-mediated reduction of antigenicity
  • Negative results in bone biopsies should be questioned because of the possible effects of decalcification or B5 fixative (fine needle aspiration or touch preparation samples are a good alternative)

Controversies

The role of quantitative reverse-transcriptase polymerase chain reaction assays measuring the level of mRNA expression is not clear at this point.

In newly diagnosed patients with node-negative, estrogen receptor–positive breast cancer, the Onco type DX assay can be used to predict the risk of recurrence in patients treated with tamoxifen. Onco type DX may be used to identify patients who are predicted to obtain the most therapeutic benefit from adjuvant tamoxifen and may not require adjuvant chemotherapy. In addition, patients with high recurrence scores appear to achieve relatively more benefit from adjuvant chemotherapy (specifically (C)MF) than from tamoxifen. There are insufficient data at present to comment on whether these conclusions generalize to hormonal therapies other than tamoxifen, or whether this assay applies to other chemotherapy regimens.

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HER2/neu Status

Indications for testing

HER2 expression and/or amplification should be evaluated in every primary invasive breast cancer either at the time of diagnosis or at the time of recurrence, principally to guide selection of trastuzumab (Herceptin, Genentech, South San Francisco, CA) in the adjuvant and/or metastatic setting. Other utilities for HER2 evaluation are also discussed separately in Table 1. [1]

Practical considerations

There is no criterion standard assay for HER2 in breast cancer. IHC and FISH are both used. Available data do not clearly demonstrate the superiority of either IHC or in situ hybridization as a predictor of benefit from anti-HER2 therapy. In 2007 ASCO and CAP published a joint guideline about HER2 testing. [12] A list of the FDA approved tests is provided in Table 4.

Table 4. FDA-Approved Tests for HER2* (Open Table in a new window)

Assay Methodology Link to Package Insert of



Manufacturer



IHC    
Hercep Test (DAKO A/S, Glostrup, Denmark) A085 polyclonal antibody http://www.dakousa.com/ prod_downloadpackageinsert.pdf?objectid=105073003
Pathway (Ventana Medical Systems, Inc., Tucson, AZ) CB11 monoclonal antibody http://www.ventanamed.com/ products/files/ScoringGuide.pdf
FISH    
PathVysion HER2 DNA probe kit (Abbott Laboratories, Illinois, USA) Hybridization of fluorescent DNA probes to HER2 gene (orange) and chromosome 17 centromere (CEP17, green). CEP17 is a centromeric probe for chromosome 17 on which the HER2 gene resides used as an internal control probe. http://www.vysis.com/ PathvysionHER2DNAProbeKit_357 93_asp
INFORM HER2/neu Probe (Ventana Medical Systems, Inc., Tucson, AZ) Hybridization of biotin-labeled DNA probe to HER2 gene and fluorescently labeled avidin http://www.ventanamed.com/catalog/ search_detail.html? id=402&categories_id=4
Chromogenic in situ hybridization    
SPOT-Light HER2CISH Kit (Invitrogen Corporation, Carlsbad, CA) Digoxigenin-tagged DNA probe detected by a fluorescent (FITC)–tagged antibody to digoxigenin followed by a horseradish peroxidase conjugated antibody to FITC and DAB http://products.invitrogen.com/ivgn/ en/US/invitrogen? cmd=catProductDetail&entryPoint=a direct&productID=840150&message Type=catProductDetail
*In addition to these commercially available tests, "home brew" IHC and FISH assays are accepted, if properly validated.

Interpretation and reporting [12]

Table 5. Interpretation and Reporting of HER2 Tests (Open Table in a new window)

  Positive Negative Equivocal
IHC



Only membranous staining is



relevant



3+



Uniform, intense membrane staining of >30% of tumor cells



0 or 1+



No staining or weak, incomplete membrane staining in any proportion of tumor cells



2+



Complete membrane staining that is either nonuniform or weak in intensity but with obvious circumferential distribution in at least 10% of cells



FISH



At least 20 non-overlapping cells in two separate areas of invasive carcinoma should be counted.



     
Tests systems with internal control probe (CEP17):



The FISH ratio (ratio of HER2 gene signals to chromosome 17 signals) is reported.



Ratio >2.2 Ratio < 1.8 Ratio 1.8-2.2
Tests systems without internal control probe:



The average number of HER2 gene copies per nucleus is reported.



>6 copies < 4 copies 4-6 copies
  Patients with positive results on IHC and/or FISH are considered eligible for trastuzumab therapy Patients with negative results on IHC and FISH are not considered eligible for trastuzumab therapy Patients with equivocal results on IHC or FISH require additional testing with the same or a different method on the same or a different sample. Eligibility for trastuzumab is controversial if all tests remain equivocal, and is up to physician judgment based on clinical variables.

No currently available assay is perfectly accurate in identifying all patients expected to benefit from anti-HER2 therapy. A combination of tests may be required on a particular specimen. The pathologist plays an important role in avoiding false-negative and false-positive results.

Quality concerns [12]

Both false-negative and false-positive results are an important concern in HER2 testing. [13, 14] HER2 testing should aim to identify all patients who would benefit from trastuzumab therapy. However, false-positive results are a significant concern because of the small risk of serious cardiac toxicity and the cost associated with trastuzumab therapy (52 weeks of trastuzumab therapy costs approximately $100,000). False-negative results can occur from fixation times that are too short or too long, decalcification and mercury-containing fixatives (B5), storage of unstained sections for more than 1 week, insufficient antigen retrieval, and nonoptimal enzymatic digestion.

Excessive antigen retrieval and accidental assessment of in situ rather than invasive lesions can lead to false-positive results. ASCO and CAP developed a joint guideline to improve the accuracy of HER2 testing. [12]

Considerations concerning pre-analytic/analytic variables are as follows:

  • Appropriate fixation: Time from tissue acquisition to fixation should be as short as possible; specimens should be fixed in neutral buffered formalin for 6-48 hours; they should be sliced at 5- to 10-mm intervals and placed in sufficient volume of neutral buffered formalin; time to fixation and duration of fixation should be recorded for each sample
  • Strict adherence to test protocols is essential
  • Appropriate controls include positive controls and negative controls (eg, cell line controls and/or tissue-based references) and an internal control (normal breast epithelium)

Considerations during pathologist interpretation of immunohistochemistry are as follows:

  • Care should be taken to make sure only the invasive component is assessed and reported, as HER2 is often overexpressed and/or amplified in in situ breast lesions
  • Controls should be reviewed; if results are not as expected, the test should be repeated
  • When possible, the test should be performed on a block with an internal control because this internal control will be an indicator of excessive antigen retrieval; if normal ducts and lobules show strong membrane staining, excessive antigen retrieval may have occurred, and repeat testing or testing via FISH should be performed
  • If cytoplasmic staining obscures membrane staining or if obscuring artifacts (edge, retraction, crush artifact) are present, the assay should be repeated or FISH should be performed
  • HER2 overexpression correlates with high tumor grade and is less common in pure tubular carcinomas, in colloid carcinomas, and in classic lobular carcinoma; [15, 16] results that are unexpected should be questioned with repeat testing or testing with an alternative method or a different sample, if indicated
  • If the results on core biopsy are questioned, repeat testing on an excision is warranted
  • Negative results on unstained sections stored for prolonged periods should be questioned; sections ideally should not be used if cut more than 6 weeks earlier
  • Negative results in bone biopsies should be questioned because of the possible effects of decalcification or B5 fixative (fine needle aspiration or touch preparation samples are a good alternative)
  • Equivocal results (2+) must be subjected to FISH testing
  • If samples were known to be fixed for periods shorter than 6 hours or longer than 48 hours, the report should qualify any negative result with a statement about fixation

As for FISH, according to ASCO/CAP guidelines, counting can be performed by a trained technologist, but the pathologist must confirm that the result (count) is correct, that invasive tumor was counted, and that the sample is surveyed for genomic heterogeneity. Other considerations are as follows:

  • Care should be taken to make sure only the invasive component is assessed; HER2 is often overexpressed and/or amplified in in situ breast lesions; corresponding H&E and/or IHC slides should be reviewed to localize the invasive carcinoma
  • Controls should be reviewed; if the controls are not as expected, the test should be repeated
  • At least 20 nonoverlapping cells in two separate areas of invasive carcinoma should be counted; counting of additional cells and/or by a different observer may be appropriate
  • Samples with nonuniform signals (weak signals in >25% of cells), high autofluorescence, poor nuclear resolution (indistinct nuclei), or high background-obscuring signal resolution (>10% of signals over cytoplasm) should be rejected
  • HER2 overexpression correlates with high tumor grade and is less common in pure tubular carcinomas, in colloid carcinomas, and in classic lobular carcinoma; [15, 16] results that are unexpected should be questioned with repeat testing or testing with an alternative method or a different sample, if indicated
  • If the results on core biopsy are questioned, repeat testing on an excision is warranted
  • Negative results on unstained sections stored for prolonged periods should be questioned; sections ideally should not be used if cut more than 6 weeks earlier
  • Negative results in bone biopsies should be questioned because of the possible effects of decalcification or B5 fixative (fine needle aspiration or touch preparation samples are a good alternative)
  • Equivocal FISH results are to be confirmed by counting additional cells or repeating the FISH test; if the FISH result remains equivocal, IHC is recommended so HER2 expression is known for the sample with true equivocal gene amplification status
  • If samples were known to be fixed for periods shorter than 6 hours or longer than 48 hours, the report should qualify any negative result with a statement about fixation

Controversies

Equivocal categories

Patients with equivocal results are a poorly studied subgroup with uncertain association of test scores to benefit from HER2-directed therapy. Equivocal IHC results (2+, complete membrane staining that is either nonuniform or weak in intensity but with obvious circumferential distribution in at least 10% of cells) are found in up to 15% of samples. Equivocal FISH results are defined by the ASCO/CAP guideline [12] as HER2/CEP17 ratio of 1.8-2.2 or average gene copy number between 4 and 6 for systems without internal control probe. It is noteworthy that patients with HER2/CEP17 ratio between 2 and 2.2 (probably < 3% of patients) were formerly eligible for treatment in the adjuvant trastuzumab trials.

Therefore, available data do not support excluding them from trastuzumab treatment. Additional repeat testing or testing with an alternative method or a different sample may be helpful. Equivocal IHC results require testing via FISH. In one study, approximately 12% of samples with 2+ IHC test results had gene amplification by FISH. [17]

Aneuploidy of chromosome 17

Polysomy 17 is seen in approximately 8% of all specimens, mostly among cases with 4-6 HER2 gene copies, the equivocal range. If polysomy 17 is defined as 3 or more CEP17 copies, most are not associated with mRNA overexpression. Most tumors with a HER2 gene copy number between 4 and 6 are also not associated with protein mRNA overexpression.

For tumors that are heterogenous for HER2 amplification on in situ hybridization (1%-2% of cases), the maximal HER2 values should be reported with a note that the tumor demonstrated heterogeneity for HER2 amplification. [18]

Discordant results

Discordant results (IHC 3+ with negative FISH result or IHC < 3+ with positive FISH result) are found in approximately 4% of patients. The available clinical data are insufficient to determine whether these represent distinct biologic subgroups with therapeutic implications or inaccurate test results.

HER2 overexpression does not equal benefit from anti-HER2 therapy. There are patients who truly overexpress HER2 but have upstream or downstream anomalies that render the interaction with trastuzumab ineffective.

Quantitative image analysis is encouraged by ASCO/CAP for cases with weak membrane staining 1-2+ to improve the consistency of interpretation.

Originally, the threshold of more than 30% of tumor cells staining for a positive (3+) IHC result was set at 10%.

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